ABSTRACT In the current funding cycle, we obtained evidence that even in non-proliferating cells the epigenome is maintained in a state of dynamic equilibrium. Given this dynamic epigenome, maintenance of a specific epigenetic state (a process for which we coined the term ?chromostasis?), and hence phenotype, over the lifespan is likely a challenge for cells. Consistent with this idea, this PO1 has also shown that age-associated epigenetic alterations can indeed by detrimental to healthy aging and longevity. One manifestation of the dynamic epigenome, the so-called ?DNA methylation clock?, uses age-associated DNA methylation changes to calculate a methylation age that is typically a strikingly accurate measure of actual chronological age. However, the extent to which this clock also reflects biological age (i.e. healthy/unhealthy aging) has not been fully defined. Recently, we co-discovered the first DNA methylation clock in the mouse and showed its slowing by multiple diverse pro-longevity interventions (Ames dwarfism, rapamycin and calorie restriction), suggesting it is indeed a biological clock. This clock largely reflects hypomethylation of genic enhancers within highly expressed genes. Hence in the renewal of this PO1, we will perform mechanistic and functional studies to determine the underlying molecular causes and consequences of the clock, and whether a biological clock can be used to predict future age-associated disease and/or mortality. The ability of rapamycin, an mTORC1 inhibitor and well known drug-based pro-longevity intervention, to slow the DNA methylation clock suggests that it may exert its pro-longevity effects, at least in part, via modulation of the epigenome. Indeed, we have obtained evidence that rapamycin stabilizes the epigenome by slowing the rate of histone eviction. We will investigate the mechanism and the extent to which this model pro- longevity intervention exerts its pro-longevity/healthy aging effects via the epigenome. Histone chaperone HIRA is a DNA replication independent chaperone that deposits histone variant H3.3 into nucleosomes, controls the dynamic epigenome of non-proliferating senescent cells and is required for chromatin integrity of these cells. Cellular senescence is a stable proliferation arrest and pro-inflammatory phenotype (the Senescence Associated Secretory Phenotype (SASP)) exhibited by viable but stressed cells. Accumulation of senescent cells in aged tissues contributes to tissue aging, in part via the pro-inflammatory SASP, a likely contributor to ?inflammaging?. Our recent studies have pointed to a role for HIRA and histone H3.3, together with the histone acetyl transferase hMOF and its histone target H4K16, in expression of the SASP. We will investigate the concerted role of HIRA, histone H3.3, hMOF and H4K16ac in expression of the SASP, in tissue aging and assess its utility as a target to suppress the SASP and promote healthy aging.